1. Field
The present disclosure relates to compositions and kits including primer sets for detecting strains of Clostridium difficile or virulent strains of Clostridium difficile and methods of detecting strains of Clostridium difficile in a sample by using the compositions or kits.
2. Description of the Related Art
Clostridium difficile is a species of Gram-positive bacteria of the genus Clostridia, which are anaerobic, spore-forming bacteria.
Clostridium difficile is the most serious cause of antibiotic-associated diarrhea and can lead to pseudomembranous colitis and a severe infection of the colon, often resulting in eradication of the normal gut flora by antibiotics. The Clostridium difficile, which naturally reside in the body, become overgrown. The overgrowth is harmful because the bacterium releases toxins that can cause bloating, constipation, and diarrhea with abdominal pain. Latent symptoms often mimic some flu-like symptoms. Discontinuation of causative antibiotic treatment is often curative.
Infections by Clostridium difficile can range in severity from asymptomatic to severe and life-threatening, especially among the elderly. People most often get infected in hospitals, nursing homes, or institutions, although infection by Clostridium difficile in the community in outpatient settings is increasing. The rate of Clostridium difficile acquisition is estimated to be 13% in patients with hospital stays of up to 2 weeks and 50% in those with hospital stays longer than 4 weeks. Frequency and severity of Clostridium difficile colitis remains high and seems to be associated with increased death rates. Early intervention and aggressive management are key factors to recovery.
Virulence factors of Clostridium difficile are toxin A, toxin B, a binary toxin, and a hypervirulent toxin. The toxin A, an enterotoxin, is encoded by tcdA genes. The toxin B, an enterotoxin, is encoded by tcdB genes. The binary toxin is encoded by cdtA and cdtB genes, and its virulence mechanism has not been identified yet. The hypervirulent toxin is related to virulent NAP1/BI/027 strains and is caused by Δ117 tcdC SNP (single nucleotide polymorphism). The emergence of a new, highly toxic strain of Clostridium difficile, resistant to fluoroquinolone antibiotics, such as Cipro (ciprofloxacin) and Levaquin (levofloxacin), said to be causing geographically dispersed outbreaks in North America was reported in 2005.
When these virulence genes are used for molecular diagnosis, there is a possibility of error occurrence due to variation of the genes and a likelihood of a false positive determination because of the variation. Thus, there is a need to detect Clostridium difficile by using a gene encoding a protein that is commonly expressed in various strains of Clostridium difficile. 
In addition, virulent Clostridium difficile induces contagious infection through its spores, and patients with the virulent strain need to be isolated. Therefore, there is a need to perform accurate and rapid diagnosis by specifically detecting virulent strains of Clostridium difficile within a short period of time.
Thus, there is a need to develop a method of primarily detecting Clostridium difficile by using a gene with little variation that is commonly expressed in various strains of Clostridium difficile, of detecting virulent Clostridium difficile strains from the detected Clostridium difficile strains with high accuracy and sensitivity within a short period of time, and of diagnosing them.